3-(2-isopentyl) and 3-isoamyl 4-acetoxybenzoyl chloride



re rates Patent 3,105,088 S-(Z-EOPENTYL) AND 3-ISGAN1YL d-ACETOXY-BENZOYL CHLGREE Herman Hoeksema, Kalamazoo Township, Kalamazoo County,lVIiclL, assignor to The Upjohn Company, Kalamazoo, Mich., a corporationof Delaware No Drawing. Filed Apr. 7, 1960, Ser. No. 20,530 3 Claims.(Cl. 260-479) This invention relates to novel compositions of matter andto a process for the preparation thereof and is particularly directed tonovel compositions of matter derived from novobiocin.

This application is a continuation-in-part of application Serial No.550,817, filed December 5, 1955, application Serial No. 576,233, filedApril 5, 1956, now Patent 2,929,- 821, and application Serial No.817,979, filed June 4, 1959, now Patent 3,026,331.

'Novobiocin, also known as streptonivicin, Antibiotic 66a, and by thetrademark Albamycin (registered U.S. Patent Ofiice), is an antibioticsubstance obtained as an elaboration product of Streptomyces niveus. Asset forth in copending US. applications of Dietz, De Boer, Smith, Bergy,and Hoeksema, Serial No. 516,742, filed June 20, 1955, now abandoned,and Serial No. 557,965, filed January 9, 1956, now abandoned, novobiocinis characterized by an optical rotation [a] =minus 63.0 degrees (c. onepercent in absolute ethanol, two decimeters); by being very soluble inWater above a pH of 9 with its solubility decreasing to about zero asthe pH decreases from 9.0 to 5.0; by being soluble in lower alkanols andacetone; by the following elemental analysis:

by a molecular weight of about 618; by an empirical 3,lli-5,088 PatentedSept. 24, 1963 formula of about C H O N by the presence of two acidicgroups: pKa 4.3, pla 9.1 in water, and pKa 5.7 and pKa 11.9 indimethylformamide; by existing in two crystal forms, form 1 melting withdecomposition between 174 and 178 degrees centigrade and form 2 meltingwith decomposition between 149 and 151 degrees centigrade, which formshave characteristic infrared absorption spectra and X-ray difiractionpatterns as set forth in the above-mentioned copending applications; byforming acid and neutral salts with both inorganic and organic bases; byultraviolet absorption maxima at 334 millimicrons in an 0.01 normalsulfuric acid solution in percent aqueous ethanol and 311 millimicron inan 0.01 normal potassium hydroxide solution in 70 percent aqueousethanol; by ultraviolet inflections at 250, 262, 282, and 304millimicrons in an 0.01 normal sulfuric acid solution in 70 percentaqueous ethanol and at 237, 255, and 287 millimicrons in an 0.01 normalpotassium hydroxide solution in 70 percent aqueous ethanol; and byactivity against a large number of gram-positive and grammegativebacteria.

It has now been found that novel compositions of matter according tothis invention are obtained by acting upon novobiocin (I) with aceticanhydride. By this procedure, the novobiocin molecule is cleavedyielding a new compound which has been identified as3-(2-isopentenyl)-4- acetoxybenzoic acid (Ai), which on alkalinehydrolysis is converted to 3-(2-isopentenyl)-4-hydrobenzoic acid (Aii),and on hydrogenation over platinum oxide to 3- isoamyl-4-acetoxybenzoicacid (Aiv). The latter compound (Aiv) on alkaline hydrolysis yields3-isoamyl-4- hydroxybenzoic acid (Av). When 3-(2-isopentenyl)-4-acetoxybenzoic acid is refluxed in the presence of concentratedhydrochloric acid, it is converted to the known2,2-dimethyl-6-chromancarboxy1ic acid (Aiii) [J .A.C.S., 65, 289-93(1943)].

These reactions are illustrated in the following chart:

CHART 1 Novobiocin C 31H3HN2 O 11 (AB Ci) 1 A020 (Aiii) H3 CHE-OH=C (Ai)H02 H01 H02 CH CH1 EtOH I 0 CH 0A0 a PtOz NaOH C2aH2aNgOm(BCi) (seeChart 2) (Ail) (Aiv) CH3 H020 /C a CH2OH=C CHs-CHz-CH H02 CH I on; OH0A0 PM /N OH 2 s a CHn-CHz-CH (Av) HO:

chloride there, is obtained 3-benzoylarnido-4,7-dihydroxy-S-methylcoumarin (Biii) which on reaction with acetic anhydrideregenerates the 2,6-dimethyl-7-acetoxy-4H-[1]-benzopyrano-[3,4-d1-oxazole (Bii). 'ihe above oxazole 5 on alkalinehydrolysis as with aqueous soduim hydroxide yields3-acetan1ido-4,7-dihydroxy-8-methylcoumarin (Bvi) which in methanolichydrochloric acid is deacetylated to 3amino-4,7-dihydroxy-8-methy1coumarin hydrochloride (Bil-1C1) whichreadily converts in Water to the free base Bi.

The above sequence of reactions is illustrated in the following chart:

CHART 2 Novobiocin (AB Oi) H01 lEtOH H (AB1) (I) /NHCO (on) I CH3EtOCgHmNOs 110- l O/\() Q/\0H3 CH3 lAczO 00-0H3 1 ll (Aii N H020 I CH:CHaCOO i O 0 CH3 CH3 H wzo (Biii) (I) NaOH NHCOGaHS HO L0 0 H H01 MeOH C3 H t (Bvi) 0 i NHCOCHa i i 0H50001 HO -0 (1)MeOH 2 H o HON 2 (Biv.HCl)o 0 (Bi) NHz-HCI NHZ CH o00- 0 NBOH 3 \0 H0 0/ 0 CH3 (5H3 acetoxy 8methylcoumarin hydrochloride (Biv.HCl) When3-arnino-4,7-dihydroxy-8-methylcoumarin (Bi) is which on alkalinehydrolysis, as with aqueous sodium hydroxide, yields3-amino-4,7-dihydroxy-8-methylcoumarin (Bi). When this compound isreacted with benzoyl treated With one normal aqueous sodium hydroxidefor several days at room temperature, or with Benedicts re- 75 agent,there is obtained upon acidification the known 2,6-

CHART 3 NHB I It has been found further that when novobiocin (ABCi) ishydrogenated over platinum oxide to dihydronovobiocin (ABCii) (seecopending application Serial No. 545,307, filed November 7, 1955, nowabandoned) and the latter refluxed with acetic anhydride, there areobtained 3- isoamyl-4-acetoxybenzoic acid (Aiv) and Compound BCi.Compound BCi also is obtained along with3(2-isopentenyl)-4-acetoxybenzoic acid (Ai) on reacting novobiocin(ABCi) with acetic anhydride. Compound BCi has the following formula:

When Compound BCi is reacted with sodium methoxide, the acetyl group(Ac) is hydrolyzed to form Compound BCiv. Acid hydrolysis of CompoundBCiv with methanolic hydrochloric acid yields 3-amino-4,7-dihydroxy-S-methylcoumarin (Bi) and methyl 3 (O)-carbamyl-4(O),5,S-trimethylpentopyranoside (Ci) of the formula having the samecarbamyl glycoside moiety as the ethyl 3(O)-carbamyl-4(O),5,5-trimethylpentopyranoside (Cii).

6 V The above sequence of reactions and the formulas of the pyranoside(Ci) areillustrated in the following chart:

CHART 4 Hz Novobiocin Dihydronovobiocin (ABCi) PtOz (AB Cii)3-(3-isopentenyl)- A020 A030 4-aeetoxybenzoie acid (A1) (see Chart 1) \lI! OOCH; 3-isoamyL4-acetoxy- I ll benzoic acid (Aiv) (see Chart 1) Ac( si5NO5) 0 (BCiv) CH3-OCH 3-amino-4,7-

dihydroxyeoumarin CHOH (Bi) (see Chart 2) O HO 0 ONE:

(7H0 CHa When methyl 3 (O)-carbamyl-4(O),5,5-trimethylpentopyranoside(Cl) is treated with boiling methanolic hydrogen chloride, the neutralnitrogen is eliminated as ammonia with the formation of a cycliccarbonate ester (Ciii) having the formula MeOC H O Treatment of thecarbonate ester with the stoichiometric amount of aqueous bariumhydroxide effects precipitation of the theoretical amount of bariumcarbonate and yields methyl 4(0),5,S-trimethylpentopyranoside (Civ)having the formula MeOC H O The methyl and ehyl3(O)carbamyl-4(O),5,5-trimethylpentopyranosides (Cifand (Cii) areneutral, ninhydrinnegative, and show only end absorption in theultraviolet.

polyol, 1,2,3,5 tetrahydroxy-4-methoxy-5-methylhexane (Cvii), CH OH-(CHOH) -CHOCH -CCH OH-CH 7 The above sequence of reactions is shown inthe following chart:

CHART 'Methyl 3 (O)-carbamyl-4 (O) ,5,5-trimethylpentopyranoside (Ci)(see Chart 3) H2O H2304 MeOkHCl (Cvi) 1/ N (Cili) H CH3 5 B (13H (IJHICHOH CHO\ ({JHOCQNH: Ba(OH)2 l /G=O CH0 CH3 CEO I I /O\ CH0 CH3 CH3 CH3BMQHV /C 1! CH3 CH3 (Civ) CH3 (I) in CHOH (IJHOH'. BaCOa CHOCHa o CH3CH3 H SO\ 1120 H t CH \1 I I M O (IEHOCH: I H CH; CH: 1 Raney nickel(Cvii) 1/ (CHOH):

CHOCH; CIEOHsOH 3 The foregoing reactions and sequences permitassignment of'the following structure for novobiocin:

The novel compounds of this invention are useful as intermediates, asbuffers, as antiseptics and disinfectants, and as antioxidants,germination inhibitors, and agents for the resolution of racemic acids.The carboxylic acids Ai, Aii, Aiii, Aiv, and Avare useful as buffers, asantiseptics and disinfectants. Also they can be converted to compoundshaving local anesthetic properties .by. first reacting them with thionylchloride to form the acid chloride and then with 'dimethylaminoethanolor with pyrrolidylalkanols according to the procedure .set forth in U.S.Patent 2,719,851. In Compounds Aii and Aiv the phenolic hydroxyl groupcan be esterified by reaction with an alkyl halide or aralkyl halidesuch as methyl, ethyl, n-propyl, isopropyl, arnyl, 2-ethylhexyl, andbenzyl chlorides, or by other known methods such as reaction withdialkyl sulfates ordiazomethane. Compound Bi is useful for theinhibition of seed germination. These ethers also are useful as buffers,antiseptics and disinfect- 8. ants, and as intermediates in theformation of local anesthetics according to U.S. Patent 2,719,851. Thepyranosides Ci, Cii, Civ, Cv, and Cvi' contain an esterifiable hydroxylgroup (Civ contains two) and can be used for resolving racemic acidssuch as d,l-mandelio and d,l-tropic acids. The resorcinol derivatives,2,6-dihydroxy-m-toluic acid and Z-methyl resorcinol, are useful asgermicides and fungicides both as such and as their mono ethers andesters such as the mono methyl ethers and mono acetates.

When 3-amino-4,7-dihydroxy-S-methylcoumarin is reduced with lithiumaluminum hydride, 2-methyl-4-(1,3-dihydroxy-Z-aminopropyl)-resourcinolis obtained. This reaction is represented as follows:

a w NH: LiAnL GH-CH-CH2 HO -O HO OH I 0 I CH3 CH3 The resultingcompound, 2-methyl 4-(l,3-dihydroxy-2- aminopropyl)-resorcinol is usefulas a fungicide, a germicide, an anthelmintic and a bronchodilator. Itsamine fluosilicate salts are useful asmothproofing agents in accordancewith U.S. Patents 1,915,334 and 2,075,359. Also in accordance with U.S.Patents 2,425,320 and 2,586,331, its thiocyanate salt can be condensedwith formaldehyde and other aldehydes to form useful picklinginhibitors. The N-alkylated analogues obtained by the lithium aluminumhydride reduction of the N-acylamino- 4,7-dihydroxy-S-methylcoumarins,such as Compounds Biii and Biv, are useful for the same purposes. The2'- methyl-4-(l ,3-dihydroxy 2 -benzylaminopropyl) resorcinol as well asother long chain alkylamino analogues such as are obtained by acylatingCompound Bi with long chain fatty acid halides, such as, lauroylchloride and oleoyl chloride, in place of the benzoyl chloride andreducing the resulting amide with lithium aluminum hydride, aresurfactants useful as detergents and Wetting agents. Their combinedwetting and germicidal and fungicidal properties make them particularlyuseful as .antiseptics and disinfectants. 'Ihe polyol Cvii can be usedin place of such substances as sorbitol, manitol, and pentaerythritol asa levigating agent in pharmaceutical compositions, as a humectant andsoftening agent for tobacco, glue, lotions and creams, and the like, andas a plasticiser. Like the prior art polyols, also the polyol Cvii isconverted to surfactants useful as detergent and wetting agents bymono-acylation with long chain fatty acid radicals. Polyol Cviimonolaurate, monopalminate, monostearate and monooleate are examples ofsurfactants which can be prepared from the compounds of this invention.The like polyacylates, for example, the distearate, can be used asointment bases. The capric acid mono ester when prepared and formulatedaccording to U.S. Patents 2,357,077 and 2,357,078 is useful as aninsecticide. The esters with drying oil acids, such as linseed oil acid,when prepared according to Ind. and Eng. Chem., 37, 809-12 (1945), areuseful as drying oils in varnish and the like.

The following examples are illustrative of the process and products ofthe present invention, but are not to be construed as limiting.

EXAMPLE 1 3-(2-Is0pentenyl)-4-Acetoxybenz0ic Acid (Ai) A solutioncontaining grams (0.162 mole) novobiocin (ABCi), one liter pyridine, and216 grams (2.14

moles) acetic anhydride was heated four hours under.

It was then chilled to five degrees centigrade' 9. extracted with ether.The ether solution was evaporated to dryness and the residue was thencrystallized from 400 milliliters ethanol and 600 milliliters of waterto yield 26 grams of 3-(2-isopentenyl)-4-acetoxybenzoic acid (Ai),melting 100-113 degrees centigrade. On recrystallization from ethanol, aproduct having the following properties was obtained:

(a) Melting point 116-120 degrees centigrade (with decomposition) (b)pKa 5.67 (50% ethanol) (c) Infrared absorption (mineral oil mull) verysimilar to acetylsalicylic acid (d) Optical rotation, none (e)Ultraviolet absorption 288 mu a=63.2 (0.01 N KOH 70% EtOH) 234 mu a=51.6(0.01 N H SO 70% EtOH) (1') Analysis The residue (51 grams) from theether extraction was recrystallized from 750 milliliters of ethanol and200 milliliters of water to yield 46.5 grams of an optically activeneutral Compound BCi having the following properties:

(a) Melting point 167-173 degrees centigrade (b) Optical rotation, [a]=minus 94.4 degrees (c. 2%

in dimethylformamide) Analysis-- Calculated for C H N O C, 56.09; H,5.73; N,

5.69. Found: C, 56.65; H, 5.44; N, 5.74.

(d) Ultraviolet absorption in 0.01 normal sulfuric acid in 70% ethanolmg a value 242 27.2 293 25.3 315 39.7 321 flex 35.0 328 flex 32.3

EXAMPLE 3 3-(2-Is0pentenyl)-4-Hydroxybenzoic Acid (Aii) A solution of0.5 gram (0.002 mole) 3-(2-isopentenyl)- 4-acetoxybenzoic acid (Ai),twenty milliliters (0.020 mole) one normal sodium hydroxide, and fiftymilliliters ethanol stood three hours at 25 degrees centigrade. It wasthen acidified with 3.3 milliliters of six normal hydrochloric acid topH 2. The solution was distilled at sixteen millimeters mercury to anaqueous concentrate, and the resulting precipitate extracted into ether.The ethereal solution was dried over sodium sulfate and evaporated todryness. The residue was crystallized from acetone and water by rapidevaporation of the acetone (crystallizing dish) to yield 350 milligrams(.0017 mole-85 percent) of 3-(2-isopentyl)-4-hyd.roxybenzoic acid (Aii)characterized as follows:

(a) Analysis Calcd. for C H O C, 69.88; H, 6.84; an wt., 206.23. Found:C, 69.89; H, 6.94; eq. wt, 208. (b) pKa 6.2, pKa 11.0, solvent 66percent alcohol 10 (c) Melting point 103-106 degrees Centigrade (d)Ultraviolet absorption- 288 m a=75.2 (0.01 normal KOH 70% EtOH) 260 ma=68.2 (0.01 normal H 70% EtOH) EXAMPLE 4 Ethyl 3-(2-Is0pentenyl)-4-Hydr0xy b'enzoate A solution of 55 grams of ethyl p-hydroxybenzoatein 200 milliliters of acetone was heated to reflux and fifty grams ofanhydrous potassium carbonate added. While gently refluxing thesolution, 33 grams of 3-methyl-3- chlorobutene (US. Patent 2,382,031)was added. The reaction mixture was heated under reflux with stirringfor three hours. The acetone was distilled and enough water added todissolve the salt and the resultant extracted with 150 milliliters ofether. To the ether extract there was added milliliters of petroleumether and the acidic components extracted with four portions of fivepercent aqueous sodium hydroxide. The solvent was then removed, the bulkby distilling and the rest by heating on a steam bath under reducedpressure for several hours. The resulting product, ethyl4-(1,1-dirnethylaliyloxy)-benzoate, was heated to boiling under reducedpressure (40 mm. Hg) until the boiling point became constant. Theproduct was then dissolved in forty milliliters of petroleum ether andextracted with five percent sodium hydroxide; The alkaline extract wasacidified with dilute sulfuric acid and the phenolic product extractedwith ether. Evaporation of the ether yielded the desired ethyl3-(2-isopentenyl)-4-hydroxybenzoate which on hydrolysis by the procedureof Example 3 gave 3-(2-isopentenyl)-4 hydroxybenzoic acid (Aii).

EXAMPLE 5 3-Is0amyl-4-Acetoxybenzoic Acid (Aiv) and Compound BCi (A) Asolution of two grams of 3-(2-isopente-nyl)-4- acetoxyhenzoic acid (Ai)in fifty milliliters absolute ethanol was hydrogenated one hour at fortypounds per square inch gauge hydrogen with one gram Adams catalyst (PtOAfter filtration, the filtrate was treated with 150 milliliters Water toyield 1.3 grams of a partially crystalline product which onrecrystallization from warm ethanol-water yielded 1.06 grams of3-isoamyl-4-ace-toxybenzoic acid (Aiv) having a melting point of 136-144degrees centigrade.

(B) A solution of two grams dihydronovobiocin (ABCii) (.0032 mole),twenty milliliters pyridine, and four grams acetic anhydride (.039 mole)was refluxed for three hours. The mixture was treated with 25milliliters water, chilled to five degrees centigrade and brought to pH1 with twenty milliliters 12 normal hydrochloric acid. The whole mixturewas extracted three times with fifty milliliters ether. The combinedextracts were Washed with 150 milliliters of water, dnied over anhydrousNa SO and evaporated to dryness. The residue was crystallized from 150milliliters of thirty percent ethanol (0.32 gram, melting point -144degrees centigrade). This material is identical to Compound Aiv.

Analysis.-Calod. for C I-1 0 C. 67.18; H, 7.25. Found: (A) C, 67.50; H,7.04. (B) C, 67.32, 67.47; H, 6.83, 6.92.

ULTRAVIOLET ABSORPTION The material which could not be extracted fromether was separated by filtration and crystallized from ethanol 11(absolute 75 milliliters) to yield 1.0 gram (melting point 155-170degrees Centigrade). It was recrystallized from milliliters 95 percentethanol to yield 0.68 gram of Compound BCi (Example 2) melting at164-173 degrees centigrade.

EXAMPLE 6 Following the procedure of Example 3, 3-isoamyl-4-acetoxybenzoic acid is hydrolyzed to 3-isoamyl-4-hydroxybenzoic acid.

' EXAMPLE 7 Compound BCiv A suspension of two grams (four millimoles) ofCompound BCi in fifty milliliters of commercial anhydrous methanol andone milliliter of one normal sodium methoxide in methanol was heatedunder reflux for thirty minutes. During this time all of the originalsolid dissolved and the product separated as white crystals. Aftercooling to room temperature, the crystals were collected,

washed with methanol and dried to yield 1.31 grams.

EXAMPLE 8 3-(2,2-Dimethyl-d-chromancarboxamido) -4,7-Dihydroxy-8-Methylcoumarin (ABi) (A) A solution of ten grams novobiocin(ABCi) (.0162 mole) in 100 milliliters absolute ethanol was heated toboiling under reflux. Following this, fifty milliliters concentratedhydrochloric acid was added to the refluxing solution :over a period ofseven minutes. (Precipitation of Compound ABi began after 45 milliliterswas added.) The mixture was heated under reflux an additional one halfhour, then cooled and filtered. The solid (6.1 grams, .0154 mole, 95percent) was recrystallized from 400 milliliters n-bntanol to yield 5.4grams.

This was then recrystallized from 1250 milliliters ethanol to yield 4.5grams of3-(2,2-dimethyl-6-chromancarboxamido)-4,7-dihydroxy-S-methylcoumarin(ABi) melting at 288-291 degrees centigrade.

Analysis.Calcd. for C H O N: C, 66.82; H, 5.35; N, 3.54. Found: C,67.52; H, 5.32; N, 3.59.

ULTRAVIOLET ABSORPTION my a EtOH KOH HQS O4 328 68. 2 X 252 84. 7 X 331661.0 X

(B) One hundred milliliters of concentrated hydrochloric acid was addedin four portions to a solution of ten grams of novobiocin in 250milliliters of boiling 95 percent ethanol over a period of about twominutes. The solution was held at the boiling point for three or fourminutes longer and3-(2,2-dimethyl-6-chromancarboxamido)-4,7-dihydroxy-S-methylcoumarin(ABi) separated as fine needles. After standing at room temperature forforty minutes and refrigerating overnight, the crystals were collected,washed with ethanol, and dried to yield 5.78 grams, melting point288-290 degrees centi grade.

EXAMPLE 9 Ethyl 3(0 -CarbamyZ-4(O ,5,5-Trimethylphent0- pyranoside (Cii)The mother liquor of Example 83 was adjusted to pH 7 by addition ofabout 190 milliliters of six normal sodiurn hydroxide and concentratedunder reduced pressure. The sodium chloride which separated was filteredoff and the filtrate was evaporated to dryness. The resulting mixture ofsyrup and sodium chloride was extracted with acetone. Concentration ofthe extract gave a semi-solid mass of gum and crystals. This wasslurried in a mixture of equal volumes of acetone and technical hexane(Skelly-Solve B); the crystals were collected, washed with fresh solventand dried to yield 316 milligrams, melting point 172176 degreescentigrade. The crude Cii crystals were recrystallized from a mixture ofequal volumes of acetone and technical hexane with 72 percent recoveryof material melting at 173-175 degrees centigrade. [a] =rninus 36degrees (c. 1 in EtOH).

Analysis.Calcd. for C I-1 N0 1 C, 50.18; H, 8.04; N, 5.32; OCH 11.79;OEt, 17.11. Found: C, 50.68; H, 8.16; N, 5.25; OCH 11.71; OEt, 17.01.

EXAMPLE 10 2,2-Dimethyl-d-Chromancarboxylic Acid (Aiii) (A) A solutioncontaining ten grams (0.25 mole) of 3(2,Z-dimethyl-6-chromancarboxamido)-4,7-dihydroxy- S-methylcoumarin(ABi), eighty milliliters pyridine, and ten milliliters (0.1 mole)acetic anhydride was heated under reflux three hours. After twomilliliters water was added to the hot solution, it was cooled to fivedegrees centigrade and acidified to pH 2 with eighty milliliters twelvenormal hydrochloric acid. The precipitate.

(fifteen grams) was crystallized in two crops, crop 1 (4.3 grams) from400 milliliters 97 percent ethanol and crop 2 (4.6 grams) from thefiltrate upon addition of 800 milliliters water. 97 percent ethanoltwice yielding 2,2-dimethyl-6-chromancarboxylic acid (Aiii) melting at184 degrees centigrade.

(B) A solution of one gram (.004 mole)3-(2-isopentenyl)-4-acetoxybenzoic acid (Ai) in ten milliliters absoluteethanol was heated to boiling. Then five milliliters twelve normalhydrochloric -acid was added and this solution refluxed one and one-halfhours. It was cooled, added to 35 milliliters of water, and extractedwith one-half volume ether. The ether was evaporated to yield an oil.Ultraviolet determinations showed the oil to have the same spectrum inacid and base, indicating an ester. The oil was dissolved in 35milliliters absolute ethanol and treated with ten milliliters (.010mole) one normal sodium hydroxide. The solution stood three days at 25degrees, was then acidified with milliliters 0.1 normal hydrochloricacid. The resulting White crystals were recrystallized from twentymilliliters fifty percent absolute ethanol to yield 0.55 gnam (67percent) of 2,2-dimethyl-6=chromancarboxylic acid (Aiii) melting at181-183 degrees centigrade.

EXAMPLE 11 Z-Methyl-S-(Z-Hydroxy-3-Methyl-4-Acet0xyphenyl)-4-Oxazolecarboxylic Acid, Delia-Lact0ne (Bii) The crop 1 crystals ofExample 10A were recrystallized twice from absolute ethanol to yield theoptically inactive 2 methyl-5-(2-hydroxy-3-methyl-4acetoxyphenyl)-4-oxazoleoarboxy\lic acid, delta-lactone-(Bii) having thefollowing properties:

(a) Melting point, 203206 degrees centigrade (b) Analysis- Calcd. for CH NO C, 61.54; H, 4.06; N, 5.13. Found: c, 61.59; H, 3.77; N, 5.06.

Crop 2 was then recrystallized from A (c) Ultraviolet absorption in 0.01normal sulfuric acid in seventy percent ethenol EXAMPLE 123-Amin0-4-Hydr0xy-7-Acetoxy-8-Methylc0umarin Hydrochloride (Biv-HCl) Asolution of two grams (7.3 millimoles) of 2-methyl-(2-hydroxy-3-methyl-4-acetoxyphenyl)-4-oxazo1ecarboxylic acid,delta-lactone (Bii), in 100 milliliters 3.5 normal methanolic hydrogenchloride and 400 milliliters methanol was heated under reflux for threehours, then stored at 24 degrees centigrade for sixteen hours. It wasconcentrated to 100 milliliters by distillation in vacuo, then chilledto minus ten degrees centigrade. The white precipitate which formed wasremoved and dried (1.50 grams), then recrystallized from 100 millilitersabsolute ethanol and 200 milliliters ether to yield 1.15 grams of 3amino-4-hydroxy-7-acetoxy-8-methylcoumarin hydrochloride (Biv-HCl) whichmelted with decomposition over the range 240310 degrees centi-grade. 'Ihis material was ninhydrin-positive, without heating, and gave a pink,fading ferric chloride test.

Analysis.-Calcd. for C H NO Cl: C, 50.09; H, 4.90; N, 4.87; Cl, 12.33;CCH 4.73; N-acetyl, 14.60; OCH;,, 00. Found: C, 49.88, 49.37; H, 5.42,5.30; N, 5.18; Cl, 11.20, 11.12; CCH 4.73; N-acetyl, 7.67; OCH 0.91.

EXAMPLE 13 3-Aceramid04,7-Dihydroxy-8-Methylcoumarin (Bvi) A solutioncontaining three grams (10.4 millimoles of 2 methyl5-(2-hydroxy-3-methyl-4-acetoxyphenyl)-4- oxazolecarboxylic acid,delta-l'actone (Bii), fifty milliliters absolute ethanol, and 100milliliters 0.6 normal sodium hydroxide was stored sixteen 'hours at 24degrees centigrade. It was acidified to pH 1 with 22 milliliters of sixnormal hydrochloric acid, then distilled in vacuo under nitrogen until alarge amount of light tan precipitate settled out. This was removed anddried yielding 2.85 grams of 3-acetamido-4,7 dihydroxyS-methyl-Coumarin, Compound Bvi. It was then recrystallized by theevaporation of acetone from a fifty percent aqueous acetone solutionyielding 2.25 grams of crystals which gradually decomposed above 230degrees centigrade and gave a negative ninhydrin test.

Analysis.Calcd. :for C H NO C, 57.83; H, 4.45; N, 5.62 (mol. wt.249.22). Found: C, 57.95, 58.03; H, 4.53, 4.12; N, 5.53, 5.53.

EXAMPLE 14 3-Amine-4,7-Dihydr0xy-8-Methylcoumarin Hydrochloride (Bi HCl)(A) A suspension of two grams (4.45 millirnoles) of Compound BViv(Example 7) in 400 milliliters of anhydrous methanol plus 100milliliters "of 3.15 normal methanolic hydrogen chloride was heatedunder reflux with frequent swirling. After about two hours all of thecrystals dissolved and the colorless solution was cooled, filtered, andconcentrated in vacuo. Two compounds crystallized and were separated byfractional crystallization from a mixture of equal volumes of methanoland ether. In this experiment, 1.19 grams of 3-amino-4,7-dihydroxy-8-methylcoumarin hydrochloride (Bi-HCl) and 0.435 gram of the methyl3(0)-carbamyl-4(O),5,5-trimethylpentopyranoside (Ci) were obtained.3-amino-4,7-d-ihydroxy- 8-methylcoumarin is less soluble in anhydrousmethanol than the methyl 3(0)-carbamyl-4(O),5,5-trimethylpentopyranoside(Ci), is amphoteric, optically inactive, gives 14 a pink color withferric chloride, and melts poorly with decomposition above 200 degreescentigrade. This product gave a positive ninhydrin reaction in the cold.

Analysis.Calcd. for C H NO Cl(243.65): C, 49.49; H, 4.15; N, 5.77; Cl,14.61. Found: C, 48.52, 48.01; H, 4.79, 5.47; N, 5.28, 5.44; Cl 12.06.

(B) A solution of one gram (four millimoles) of 3-acetamido-4,7-dihydroxy-8-methylcoumarin (Bvi) in fifty milliliters of3.5 normal methanolic hydrogen chloride and 200 milliliters anhydrousmethanol was heated under reflux for two hours and stored at 24 degreescentigrade for sixteen hours. The solution was concentrated bydistillation in vacuo to a fifty-milliliter volume, diluted with 200milliliters ether and cooled to four degrees centigrade. The whitecrystals of 3-amino-4,7-dihydroxy-8-methylcoumarin hydrochloride(Bi-HCl) which separated were removed and dried (0.74) gram). Thismaterial retained its birefringence to 140 degrees centigrade butdecomposed without rnelting upon heating to 300 degrees centigrade. Thismaterial was ninhydrin-positive in the cold and gave a pink ferricchloride test.

EXAMPLE 15 3-Amino-4,7-Dihydroxy-8-Methylcoumarin (Bi) (A)Recrystallization of the hydrochloride of Example 14 from water yieldedthe free *base or Zwitter-ion compound,3-amino-4,7-dihydroxy-8-methylcoumarin (Bi).

(B) A -milligram quantity of 3-amino-4-hydroxy-7-acetoxy-8-methylcoumarin hydrochloride (Biv-HOI) was recrystallizedfrom hot thirty percent aqueous ethanol to give3-amino-4,7-dihydroxy-8-methylcoumarin (Bi). The dried crystallineproduct was birefringent to 190 degrees centigrade, but decomposedwithout melting upon heating to 300 degrees centigrade. The material wasninhydrin-posit-ive in the cold, and gave a pink, fading ferric chloridetest.

Analysis.Calcd. for C H NO C, 57.99; H, 4.38; N, 6.77; Cl, 00. Found:(A) C, 57.69; H, 4.32; N, 6.25. (B) C, 56.66, 56.37; H, 5.15, 4.79; N,6.20; Cl, 0.10.

EXAMPLE 16 Synthesis of 3-Amin0-4,7-Dihydroxy-8-Methylcoumarin (A)4,7-DIHYDROXYJ8-METHYLCOUMABIN Dry hydrogen chloride was passed for twohours into a chilled mixture of 18.6 grams (0.1 5 mole) ofZ-methylresorcinol, 18.6 grams (0.165 mole) of ethyl cyanoacetate, and15 grams of fused, powdered Zinc chloride in milliliters of anhydrousether. The mixture was allowed to stand overnight at room temperatureand the ether was decanted. A large volume of water was added to theresidue and the intermediate 7-hydroxy-8-methyl-4-imino-3,4-dihydrocoumarin was obtained as a solid which was isolatedby filtration. The moist solid was heated for several hours withapproximately ten times its weight of fifty percent sulfuric acid.Dilution with a large volume of Water precipitated4,7-dihydroxy-8-methylcoumarin which was collected by filtration anddired.

(B) 3-AMINO+1,TDIHYDROXY-S-HETHXLCOUDLARIN Three grams of4,7-dihydro-8-methylcoumarin was nitrated by the procedure of Link etal., J.A.C.S., 67, 99 (1945), and the resulting3-nitro-4,7-dihydroxy-8-methylcournarin was catalytically hydrogenated(ibid.) to 3 amino-4,7-dihydroxy 8 methylcoumarin. A melting point of amixture of this product and that obtained by degradation of theantibiotic exhibited no depression.

EXAMPLE 17 3-Benzamid0-4,7-Dihydroxy-8-Methylcoumarin (Bz'ii) Benzoylchloride (0.6 milliliter) was added with vigorous shaking in three equalportions to a solution of 0.46 gram of3-amino-4,7-dihydroxy-8-methylcoumarin (Bi) dissolved in thirtymilliliters of one normal sodium hydroxide. The reaction mixture wascooled in an ice bath. After two hours, the solution was filtered freeof a trace of alkali-insoluble material and acidified with two normalhydrochloric acid. The orange precipitate which formed was filtered ofi,washed thoroughly with water and dried in a vacuum desiccator. The crudeproduct was extracted with four 30-40-milliliter portions of boilingtechnical hexane (Skellysolve B) to remove benzoic acid. The materialwhich failed to dissolve in technical hexane was recrystallized from 95percent aqueous ethanol using Darco G-60 for clecolorization, yielding0.398 gram of 3- benzamido-4,7dihydroxy-8-methylcoumarin (Biii) as verypale yellow crystals, melting point 309-310 degrees centigrade. Foranalysis, a portion of this material was recrystallized again from 95percent aqueous ethanol.

AnaZysis.-Calcd. for C H NO (311.28): C, 65.59; H, 4.21; N, 4.50. Found:C, 65.63, 65.77; H, 4.51, 4.19; N, 4.42.

Following the procedure of Example 17 substituting the benzoyl chlorideby propionyl chloride, lauroyl chloride, and oleoyl chloride, there areobtained the corresponding N-alkanoylamid-es, namely,3-propionamido-4,7-dihydroxy-S-methylcoumarin, 3l-auramido-4,7-dihydroxy-8- methylcoumarin, and3-oleamido-4,7-dihydroxy-8-methylcournarin.

EXAMPLE 18 2-Methyl-5-(2-Hydr0xy-3-Methyl 4-Acetoxyphenyl)-4-Oxazolecarboxylic Acid, Delta-Lactone (Bii) A solution of 515 milligramsof the benzoyl derivative of Example 17 in 45 milliliters of pyridineand fifteen milliliters of acetic anhydride was allowed to stand at roomtemperature for one hour and was then heated under reflux for one to twohours. After cooling, water and solid potassium bicarbonate were addedand the solution was taken to dryness in vacuo. The residue was taken upinwater and chloroform. After shaking, the layers were separated, andthe aqueous phase was extracted with several portions of chloroform. Thecombined extracts were evaporated to dryness and the residue wasrecrystallized from ethanol and water to yield 100 milligrams ofZ-methyl-5 (2-hydroxy-3-methyl-4-acetoxyphenol) 4 oxazolecarboxylicacid, delta-lactone (Bii) nearly colorless crystals which melted at201-203 degrees centigrade.

Analysis.-Calcd. for C H NO (273.24): C, 61.54; H, 4.07; N, 5.13. Found:C, 61.84; H, 3.80; N, 5.12.

EXAMPLE 19 4,7-Dihydroxy-3- [4-Hydroxy-3-(2-Is0pentenyl)Benzamido]-8-Methylc0umarin (ABii) To ten grams of novohioein (0.016mole) in 100 milliliters of absolute ethanol, there was added 0.5 gram(0.0064 mole) of acetyl chloride as a catalyst. The reaction mixture washeated under reflux for two hours and cooled. With stirring there wasslowly added 300 milliliters of water. After filtering and washing withcold anhydrous ethanol, there was obtained six grams of 4,7 dihydroxy 3[4 hydroxy 3 (2 isopentenyl)- benzamido1-8-methylcoumarin (ABii) havinga melting point of 167-175 degrees centigrade. This compound hasantibacterial activity against such organisms as Strep- :tococcushemolyticus, Bacillus subtilus, and Staphylococrus albus.

On treatment of this compound with acetic anhydride by the procedure ofExamples A and 11, there was obtained Compounds Bii and Ai. On treatmentwith ethanol and concentrated hydrochloric acid by the procedure ofExample 8A, there was obtained Compound ABi. On hydrogenation overplatinum oxide by the procedure of Example 5A, there was obtained4,7-dihydroxy 3 (4 hydroxy 3 isoamylbenzamido) 8- methylcoumarin(ABiii). Compound A-Biii can also be obtained by treatingdihydronovobiocin (ABCii) with ethanol and a catalytic amount of acetylchloride by the procedure of this example. Compound ABiii has the sameantibacterial activity as Compound ABii. On reacting Compound ABiii withacetic anhydride by the procedure of Examples 10A and 11, there wasobtained Compounds Bii and Aiv.

The sequence of reactions given in this example are shown in thefollowing chart:

CHART 6 Novoblocin (ABOi) EtOH 101130001 H ii) l /OH:

NHC O CH2CH=C\ 1 0 0 on CH3 \HCl A00: nron\\ /\(.AD H2 P1302 l! \l(Bii)\ (A (Aiv) Dihydronovobiocin Ha ACO: /Et02 H 0 CH3 1 NHOO CH2CHzGgCH3 (ABiii) HO o 0 on I OH;

ExAMPLE 20 4-Acetoxy-3-(Z-Isopropenyl)-Benz0yl Chloride (Avi) To tengrams (0.04 mole) of Compound Ai (Example 1) was added twentymilliliters of thionyl chloride. The mixture was stirred ten minutes ateighty degrees centigrade, then cooled and diluted with thirtymilliliters of water. The oily precipitate was extracted into fiftymilliliters of ether. On evaporation of the ether there was obtained9.8; grams of 4-acetoxy-3-(2-isopropenyl)-be-nzoyl chloride.

EXAMPLE 21 4-Acet0xy-3-Isoamylbenz0yl Chloride' (Aviz') On substitutingCompound Aiv (Example 5) for Compound Ai in the procedure of Example 20,there was obtained 4-acetoxy-3-isoa-mylbenzoyl chloride.

EXAMPLE 22 4,7-Dihydr0xy-3-[4-Hydroxy-3-(2-Is0pentenyl)- Benzamz'do]-8-Methylcoumarin (ABii) On substituting Compound Avi (Example 20) forthe benzoyl chloride of Example 17, there was obtained 4,7 dihydroxy 3[4 hydroxy 3 (2 isopentenyD- benzamido] -8-methylcoumarin.

1 7 EXAMPLE 23 4,7-Dihydrxy-3- (4-Hydr0xy-3-Isoamylbenzamido) -8- Methylcoumarin (ABiii) On substituting Compound Avii (Example 21) for thebenzoyl chloride of Example 17, there was obtained 4,7- dihydroxy 3 (4hydroxy -,3 isoamylbenzamido) 8- methylcoumarin.

EXAMPLE 24 Methyl 3 (O)-Carbamyl-4 (O),5,5-Trimethylpentopyranoside (Ci)The methyl pyranoside Ci produced in Example 14A was recrystallized froma mixture of equal volumes of acetone and technical hexane (SkellysolveB) to give colorless plates which are neutral, ninhydrin-negative, showonly end absorption in the ultraviolet, melt at 197- 198 degreescentigrade and are optically active.

[a] =minus 24.7 degrees (c. 1 in 95 percent 'EtOH).

Analysis.-Calcd. for C H NO (249.29): C, 48.19; H, 7.68; N, 5.62. Found:C, 48.11; H, 7.71; N, 5.61.

EXAMPLE 25 Carbonate Ester Ciii A solution of 1.03 grams (fourmillimoles) of methyl 3(O)-carbamyl-4(O),5,5-trimethylpentopyranoside(Ci) in 175 milliliters of absolute methanol and forty milliliters ofthree normal methanolic hydrogen chloride was heated under reflux for3.5 hours. The solution was concentrated to dryness in vacuo. Theresidue was slurried in ten to twelve milliliters of acetone and 98.4milligrams of insoluble ammonium chloride removed. The filtrate wasevaporated to about five milliliters under nitrogen jet and 245.2milligrams of unreacted methyl pyranoside Ci recovered. The filtrate wasconcentrated under nitrogen to dryness. The remaining hydrogen chloridewas dispelled by vacuum drying and the residue was extracted withfifteen milliliters of hot water. On cooling, a crop of colorlesscrystals was obtained, 109 milligrams, melting point 122-128 degreescentigrade. The crude crystals were purified by sublimation at about 120degrees centigrade and 20-25 millimeters mercury. The sublimate meltedat 132-1325 degrees centigrade.

Analycis.--Calcd. for C H O C, 5.712 H; H, 6.95; 2OCH 26.73. Found: C,51.97; H, 6.91; OCH 27.59.

EXAMPLE 26 Methyl 4 (O),5,5-Trimethylpentopyranoside (Civ) Two grams(about eight millirnoles) of the methyl pyranoside Ci of Example 24 wasdissolved in 75 milliliters of water by warming. The solution was cooledto room temperature and 55 milliliters of saturated barium droxidesolution Was added. After standing for sixteen hours at room temperaturein a flask protected from atmospheric carbon dioxide with an Ascarite(NaOH on asbestos) tube, the precipitate of barium carbonate was removedby filtration. The excess barium hydroxide was neutralized with carbondioxide (Dry Ice) and again the precipitate was removed by filtration.The filtrate was taken to dryness in vacuo and the residue dissolved inether. The solution was filtered free of a small amount of bariumcarbonate and evaporated to yield 1.608 grams (97%) of a colorless oilwhich crystallized on standing. After recrystallization from boilingtechnical hexane (Skellysolve B), the product melted at 65-70 degreesand had a specific rotation [m] =minus 39 degrees (c. 1.0 in 0.5 normalsulfuric acid). The compound consumes one mole :of periodate rapidlywithout the formation otE acidic products 101' =formaldehyde.

Analysis.Calcd. for C H O C, 52.41; H, 8.80; OCH 30.0. Found: C, 52.82;H, 8.55; OCH 26.8.

Methyl 4(0),5,S-trimethylpentopyranoside (Civ) can also be made from thecarbonate ester Ciii of Example 25 by the same procedure with thismodification: the reaction with barium hydroxide requires only one hourinstead of sixteen hours.

EXAMPLE 27 4(0) ,5,S-Trimethylaldopentose (Cv) barium carbonate wasadded to neutralize the sulfuric acid. The precipitate of barium sulfateand barium carbonate was removed by filtration and the neutral filtrateconcentrated to dryness in vacuo. The residue was taken up in ethanol,the solution filtered free of insoluble salts, and evaporated to yield1.07 :grams (96%) of a colorless oil which crystallized on drying in avacuum desiccator. After recrystallization fir-om ethyl acetate, thecrystals melted at 128-130 degrees Centigrade with a specific rotationwith a specific rotation [a] =plus 38 degrees (c. 1.0 in 0.5 normalsulfuric acid). The compound consumes two moles of periodate with theionmation of two moles of formic acid but no formaldehyde.

Analysis.Calcd. for C H O C, 49.98; H, 8.39; OCH 16.1. Found: C, 49.71;H, 8.50; OOH 14.95.

EXAMPLE 28 3(0) -Carbamyl-4(0),5,5-Trimethylpentopyranose (Cvi) Asolution of 300 milligrams of methyl 3 (O)-carbamyl-4(0),5,5-trimethylpentopyranoside (Ci) in thirty milliliters of 0.5normal sulfuric acid was heated at eighty degrees until the observedrotation reached a constant value (50-60 minutes). The solution wascooled quickly to room temperature and neutralized with'an excess ofbarium carbonate. After removing the insoluble salts, the solution wasconcentrated to dryness and the residue taken up in ethanol. The smallamount of insoluble salts was removed by filtration and the filtrateevaporated to dryness to yield 290 milligrams of a colorless glass. Theproduct consumed one mole of periodate with the formation of one mole offormic acid.

Analysis.Calcd. for C H NO C, 45.95; H, 7.29; N, 5.96. Found: C, 45.95;H, 7.66; N, 5.92.

By heating 3(O)-carbarnyl-4(0),5,5-trimethylpyranose (Cvi) in an alcoholsaturated with dry hydrogen chloride, the corresponding pyranoside isobtained. For example, from such lower alkanols as methanol, ethanol,isopropanol, n-propanol, n-butanol, 2-ethylhexanol, and the like, thereare obtained methyl, ethyl, isopropyl, n-propyl, n-butyl, and2-ethylhexyl 3(O)-carba-myl-4(O),5,5-trimethylpyranosides. By treatingthese pyranosides by the procedure of Example 26, ethyl, isopropyl,n-propyl, nbutyl, and 2-ethylhexyl 4(0),5,S-trimethylpentopyranosidesare obtained. The corresponding carbonate esters are obtained by theprocedure of Example 25.

EXAMPLE 29 1,2,3,5-Tetrahydroxy4-Methoxy-4-Methylhexane (Cvii) Asolution of 400 milligrams of 4(O),5,5-trimethylaldopentose (Cv) in 25milliliters of percent aqueous ethanol was hydrogenated at forty poundsper square 'inch gauge hydrogen pressure in the presence of milligramsof platinum oxide catalyst for sixteen hours. The solution was filteredfree of catalyst and evaporated to yield 405 milligrams of a colorlessviscous oil which consumed two moles of period-ate within one hour withthe production of one mole of formaldehyde and one mole of formic acid.The formaldehyde was isolated as the dimedon derivative and the formicacid was identified by potentiometric titration.

It is to be understood that the invention is not to be limited to theexact details of operation or exact compounds shown and described, asobvious modifications and equivalents will be apparent to one skilled inthe art,

19 20 I and the invention is therefore to be limited only by theisopentenyl and isoamyl and R is selected from the class scope of theappended claims. consisting of hydrogen and acetyl.

I claim: 2. 4-acetoxy-34(2-isopentenyl)benzoyl chloride. 1. A compoundhaving the formula: 3. 4-acetoxy-3-isoamylbenzoy1 chloride.

. 5 7 0001 References Cited in the file of this patent UNITED STATESPATENTS R 2,696,498 Hooh et a1. Dec. 7, 1943 V 10 OTHER REFERENCESJ.A.C.S., vol. 65, pp. 289-93, 1943.

wherein R is selected (from the class consisting of 2-

1. A COMPOUND HAVIG THE FORMULA: